Studies of Protein Phase Separation Using Leishmania\nKinetoplastid Membrane Protein-11

Despite\nthe significant understanding of phase separation in proteins\nwith intrinsically disordered regions, a considerable percentage of\nproteins without such regions also undergo phase separation, presenting\nan intriguing area for ongoing research across all kingdoms of life.\nUsing a combination of spectroscopic and microscopic techniques, we\nreport here for the first time that a low temperature and low pH can\ntrigger the liquid–liquid phase separation (LLPS) of a parasitic\nprotein, kinetoplastid membrane protein-11 (KMP-11). Electrostatic\nand hydrophobic forces are found to be essential for the formation\nand stability of phase-separated protein assemblies. We show further\nthat the increase in the ionic strength beyond a threshold decreases\nthe interchain electrostatic interactions acting between the alternate\ncharged blocks, altering the propensity for phase separation. More\ninterestingly, the addition of cholesterol inhibits LLPS by engaging\nthe cholesterol recognition amino acid consensus (CRAC)-like domains\npresent in the protein. This was further confirmed using a CRAC-deleted\nmutant with perturbed cholesterol binding, which did not undergo LLPS.